Identification of small RNAs We have carried out several different systematic screens for small regulatory RNA genes in E. coli. These screens are all applicable to other organisms. One approach based on computer searches of intergenic regions for extended regions of conservation among closely related species led to the identification of 17 conserved small RNAs. Another screen for small RNAs that coimmunoprecipitate with the RNA binding protein Hfq allowed us to detect six less well conserved RNAs. A third approach of size fraction of total RNA followed by linker ligation and cDNA synthesis resulted in the identification of still other small RNAs. We have recently obtained tiled microarrays which provide coverage of the whole E. coli genome and are using these arrays to extend our identification of the small RNAs. Characterization of specific small RNAs An expanding focus of the group has been to elucidate the functions of the small RNAs in E. coli. We previously showed that the OxyS RNA, whose expression is induced in response to oxidative stress, acts to repress translation by basepairing with target mRNAs. OxyS RNA action is dependent on the Sm-like Hfq protein, which functions as a chaperone to facilitate OxyS RNA basepairing with its target mRNAs. We also discovered that the abundant 6S RNA binds and modifies RNA polymerase. In addition, we elucidated the functions of the MicC RNA and the GadY RNA, which also bind Hfq and act by basepairing. We found the MicC RNA represses translation of the OmpC outer membrane porin. Interestingly, under most conditions, the MicC RNA shows expression opposite that of the MicF RNA, which represses expression of the OmpF porin. Basepairing between the GadY RNA and the 3-untranslated region (3 UTR) of the gadX mRNA encoded opposite gadY leads to increased levels of the gadX mRNA and GadX protein. Increased GadX levels in turn result in increased expression of the acid-response genes controlled by the GadX transcription factor. In one recent study we characterized a small RNA (SymR), which is encoded in cis to an SOS-induced gene whose product shows homology to the antitoxin MazE (SymE). We showed that synthesis of the SymE protein is tightly repressed at multiple levels by the LexA repressor, the SymR RNA and the Lon protease. SymE co-purifies with ribosomes and overproduction of the protein leads to cell growth inhibition, decreased protein synthesis and increased RNA degradation. These properties are shared with several RNA endonuclease toxins of the toxin-antitoxin modules, and we reported that the SymE protein represents evolution of a toxin from the AbrB fold, whose representatives are typically antitoxins. We suggest that SymE promotion of RNA cleavage may be important for the recycling of RNAs damaged under SOS-inducing conditions. In another recent study, we characterized the Sib RNAs, which are encoded by five repeats in Escherichia coli K-12, though the number of repeats varies among E. coli strains. All five Sib RNAs in E. coli K-12 are expressed, and no phenotype was observed for a five sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18-19 amino acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by co-expression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell. Studies to further characterize the OxyS, GadY and the Sib RNAs and to elucidate the roles of other newly-discovered small RNAs are ongoing.